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At least 343 records · Page 19Linked to original sources
PubMed1999

Dcdc42 acts in TGF-beta signaling during Drosophila morphogenesis: distinct roles for the Drac1/JNK and Dcdc42/TGF-beta cascades in cytoskeletal regulation.

During Drosophila embryogenesis the two halves of the lateral epidermis migrate dorsally over a surface of flattened cells, the amnioserosa, and meet at the dorsal midline in order to form the continuous sheet of the larval epidermis. During this process of epithelial migration, known as dorsal closure, signaling from a Jun-amino-terminal-kinase cascade causes the production of the secreted transforming-growth-factor-beta-like ligand, Decapentaplegic. Binding of Decapentaplegic to the putative transforming-growth-factor-beta-like receptors Thickveins and Punt activates a transforming-growth-factor-beta-like pathway that is also required for dorsal closure. Mutations in genes involved in either the Jun-amino-terminal-kinase cascade or the transforming-growth-factor-beta-like signaling pathway can disrupt dorsal closure. Our findings show that although these pathways are linked they are not equivalent in function. Signaling by the Jun-amino-terminal-kinase cascade may be initiated by the small Ras-like GTPase Drac1 and acts to assemble the cytoskeleton and specify the identity of the first row of cells of the epidermis prior to the onset of dorsal closure. Signaling in the transforming-growth-factor-beta-like pathway is mediated by Dcdc42, and acts during the closure process to control the mechanics of the migration process, most likely via its putative effector kinase DPAK.

Animals↗
PubMed2006

Oligomerizing potential of a focal adhesion LIM protein Hic-5 organizing a nuclear-cytoplasmic shuttling complex.

Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacted with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affected the subcellular distribution of PINCH but directed its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments.

Active Transport, Cell Nucleus↗
PubMed2002

How signaling proteins integrate multiple inputs: a comparison of N-WASP and Cdk2.

Signal transduction proteins that can integrate multiple upstream signals play a critical role in the complex regulatory circuits that control cellular behavior. The two signaling node proteins cyclin-dependent kinase 2 and the actin regulator neuronal Wiskott-Aldrich syndrome protein have qualitatively similar signaling properties. Recent studies, however, reveal that these proteins utilize distinct mechanisms of signal integration, leading to subtle but important quantitative differences in behavior.

Animals↗
PubMed2005

cDNA cloning, sequence analysis of the porcine LIM and cysteine-rich domain 1 gene.

LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

Amino Acid Sequence↗
PubMed1992

Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells.

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.

Alkaloids↗
PubMed2000

Cloning and chromosomal localization of a human class III myosin.

Two class III myosins have been identified to date: NINAC from Drosophila melanogaster and MyoIII(Lim) from Limulus polyphemus. Both have N-terminal kinase domains and are expressed exclusively in photoreceptors. Mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival. We report the cloning and chromosomal localization of a human class III myosin, MYO3A, from retina and a retinal pigment epithelial cell line. Human MYO3A (which we will refer to simply as MYO3A) possesses an N-terminal kinase domain and three consensus calmodulin-binding (IQ) motifs, two in the neck and one in the tail domain. We detected two MYO3A splice variants differing by 52 amino acids near the kinase/myosin junction. On Northern blots, MYO3A probes detected a 6. 5-kb transcript in human and monkey retina, in a cultured human RPE cell line (RPE-19), and at much lower levels in human pancreas. A somatic hybrid panel PCR screen localized MYO3A to human chromosome 10, and a radiation hybrid screen further localized it proximal to marker D10S197, which is located at 10p11.1 on the human cytogenetic map. Since mutations in NINAC have been shown to alter the photoresponse and compromise photoreceptor survival, the human homologue MYO3A may also play a role in photoreceptor function and/or maintenance.

Aged↗
PubMed2002

Developmental expression of serum response factor in the rat central nervous system.

Serum response factor (SRF), a transcription factor known to be essential for early embryonic development as well as post-natal regulation of both cellular proliferation and myogenic differentiation, is expressed broadly in neurons within the adult mammalian central nervous system (CNS). The function of SRF within the developing CNS is not well established, but it is likely to play an important role in neuraxial development and neuronal function, since many of its known target genes (e.g., c-fos) and transcriptional partners (e.g., Elk-1) are also highly expressed in neurons. Immunohistochemical survey of the post-natal developing rat brain revealed a progressive increase in SRF immunoreactivity in neurons of the cerebral and cerebellar cortices, and in selective subcortical regions from birth (P0) through post-natal day 28 (P28). SRF immunoreactivity stabilized from P28 into adulthood. A few loci, such as the nucleus of cranial nerve VII, showed the reverse expression pattern (strong immunoreactivity at P0-P7, declining by P28). The developmental expression pattern of SRF overlaps significantly with that of myotonic dystrophy protein kinase, a potential upstream regulator, and of the LIM-only genes Lmo1, Lmo2 and Lmo3, whose products belong to a family of proteins known to be strong positive regulators of SRF's transcriptional activity. These data suggest that SRF has a significant function in the early post-natal development of the CNS.

Age Factors↗
PubMed2001

Role of LIM domains in mediating signaling protein interactions.

LIM domains are double zinc-finger motifs that mediate protein interactions between transcription factors, cytoskeletal and signaling proteins. This review outlines the protein interactions mediated by LIM domains with signaling proteins including tyrosine and serine/threonine kinases and phosphatases.

Actins↗
PubMed2005

The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A.

Previously, we showed PAK-PIX-GIT targets and regulates focal adhesions; here, we uncover a different function for the complex at the centrosome. Active PAK1 is particularly evident in mitosis and phosphorylates the centrosomal adaptor GIT1 on serine 517. Interestingly, direct centrosome targeting activates the kinase via a process not requiring Rho GTPases; excision of the centrosome prevents this activation. Once activated, PAK1 dissociates from PIX/GIT but can bind to and phosphorylate the important centrosomal kinase Aurora-A. PAK1 promotes phosphorylation of Aurora-A on Thr288 and Ser342, which are key sites for kinase activation in mitosis. In vivo PAK activation causes an accumulation of activated Aurora-A; conversely, when betaPIX is depleted or PAK is inhibited, there is a delay in centrosome maturation. These observations may underlie reported effects of active PAK on cells, including histone H3 phosphorylation, alterations in centrosome number, and progression through mitosis.

Animals↗
PubMed2006

Serotonin-, protein kinase C-, and Hic-5-associated redistribution of the platelet serotonin transporter.

Emerging data indicate the existence of multiple regulatory processes supporting serotonin (5HT) transporter (SERT) capacity including regulated trafficking and catalytic activation, influenced by post-translational modifications and transporter-associated proteins. In the present study, using differential extraction and sedimentation procedures optimized for the purification of cytoskeletal and membrane-skeletal associated proteins, we analyze SERT localization in platelets. We find that most of the plasma membrane SERT is associated with the membrane skeleton. This association can be enhanced by both transporter activation and 5HT2A receptor activation. Inactivation of transport activity by phorbol ester treatment of intact platelets relocates SERT to the cytoskeleton fraction, consequently leading to transporter internalization. The translocation of SERT between these compartments is correlated with changes in the interaction with the LIM domain adaptor protein Hic-5. Co-immunoprecipitation and uptake activity studies suggest that Hic-5 is a determinant of transporter inactivation and relocation to a compartment subserving endocytic regulation. Associations of SERT with Hic-5 are evident in brain synaptosomes, suggesting the existence of parallel mechanisms operating to regulate SERT at serotonergic synapses.

Animals↗
PubMed1999

Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene.

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nucleotide sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5' and 3' flanking regions of the pseudogene and the poly(A) tract in the 3' end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.

Adenylate Kinase↗
PubMed2002

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling.

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.

Amino Acid Sequence↗
PubMed2005

Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal.

Survival of near-freezing body temperatures and reduced blood flow during hibernation is likely the result of changes in the expression of specific genes. In this study, we described a comprehensive survey of mRNAs in the heart of the thirteen-lined ground squirrel (Spermophilus tridecemlineatus) before and during hibernation. The heart was chosen for this study because it is a contractile organ that must continue to work despite body temperatures of 5 degrees C and the lack of food for periods of 5-6 mo. We used a digital gene expression assay involving high-throughput sequencing of directional cDNA libraries from hearts of active and hibernating ground squirrels to determine the identity and frequency of 3,532 expressed sequence tags (ESTs). Statistical analysis of the active and hibernating heart expression profile indicated the differential regulation of 48 genes based on a P < or = 0.03 threshold. Several of the differentially expressed genes identified in this screen encode proteins that likely account for uninterrupted cardiac function during hibernation, including those involved in metabolism, contractility, Ca2+ handling, and low-temperature catalysis. A sampling of genes showing higher expression during hibernation includes phosphofructokinase, pancreatic triacylglycerol lipase, pyruvate dehydrogenase kinase 4 (PDK4), aldolase A, sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), titin, and four-and-a-half LIM domains protein 2 (FHL2). Genes showing reduced levels of expression during hibernation include cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), troponin C, phospholamban, Ca2+/calmodulin-dependent protein kinase II (CaMKII), calmodulin, and four subunits of cytochrome c oxidase.

Adaptation, Physiological↗
PubMed1998

Interaction of Hic-5, A senescence-related protein, with focal adhesion kinase.

Hydrogen peroxide-inducible clone (Hic)-5 is induced during the senescent process in human fibroblasts, and the overexpression of Hic-5 induces a senescence-like phenotype. Structurally, Hic-5 and paxillin, a 68-kDa cytoskeletal protein, share homology such as the LD motifs in the N-terminal half and the LIM domains in the C-terminal half. Here we show that Hic-5 binds to focal adhesion kinase (FAK) by its N-terminal domain, and is localized to focal adhesions by its C-terminal LIM domains. However, Hic-5 is not tyrosine phosphorylated either by the coexpressed FAK in COS cells or by integrin stimulation in 293T cells. Furthermore, overexpression of Hic-5 results in a decreased tyrosine phosphorylation of paxillin. These findings suggest that putative functions of Hic-5 are the recruitment of FAK to focal adhesions and a competitive inhibition of tyrosine phosphorylation of paxillin.

Amino Acid Sequence↗
PubMed1990

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

Amino Acid Sequence↗
PubMed1999

Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain.

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.

Animals↗
PubMed2001

Treble clef finger--a functionally diverse zinc-binding structural motif.

Detection of similarity is particularly difficult for small proteins and thus connections between many of them remain unnoticed. Structure and sequence analysis of several metal-binding proteins reveals unexpected similarities in structural domains classified as different protein folds in SCOP and suggests unification of seven folds that belong to two protein classes. The common motif, termed treble clef finger in this study, forms the protein structural core and is 25-45 residues long. The treble clef motif is assembled around the central zinc ion and consists of a zinc knuckle, loop, beta-hairpin and an alpha-helix. The knuckle and the first turn of the helix each incorporate two zinc ligands. Treble clef domains constitute the core of many structures such as ribosomal proteins L24E and S14, RING fingers, protein kinase cysteine-rich domains, nuclear receptor-like fingers, LIM domains, phosphatidylinositol-3-phosphate-binding domains and His-Me finger endonucleases. The treble clef finger is a uniquely versatile motif adaptable for various functions. This small domain with a 25 residue structural core can accommodate eight different metal-binding sites and can have many types of functions from binding of nucleic acids, proteins and small molecules, to catalysis of phosphodiester bond hydrolysis. Treble clef motifs are frequently incorporated in larger structures or occur in doublets. Present analysis suggests that the treble clef motif defines a distinct structural fold found in proteins with diverse functional properties and forms one of the major zinc finger groups.

ADP-Ribosylation Factors↗
PubMed2002

Nuclear translocation of cell adhesion kinase beta/proline-rich tyrosine kinase 2.

Cell adhesion kinase beta (CAKbeta/PYK2) is a protein-tyrosine kinase of the focal adhesion kinase (FAK) family. Whereas FAK predominantly localizes at focal adhesions, CAK beta localizes at the perinuclear region in fibroblasts. Here we expressed in cultured cells two point mutants of CAKbeta, P717A and P859A, each of which had lost one of its two PXXP motifs, the ligand sequence for SH3 domains, found at the CAKbeta C-terminal region. We observed a remarkable change in the subcellular distribution of the P859A mutant; while that of the P717A mutant was the same as the wild type. The P859A mutant localized exclusively in the cell nucleus in all cell lines examined. Wild-type CAKbeta also accumulated in the nucleus when cells were treated with an inhibitor of the nuclear export of proteins. These results indicate that CAK beta shuttles between the cytoplasm and the nucleus. On nuclear accumulation of P859A-CAKbeta, a CAKbeta-binding protein, Hic-5, also accumulated in the nucleus. P859A-CAKbeta and co-expressed Hic-5 formed nuclear speckles, in which one other CAK beta-binding protein, p130(Cas), was also concentrated. These findings on nuclear translocation of CAK beta imply that CAKbeta may regulate nuclear processes such as transcription, particularly because Hic-5 was recently shown to be a coactivator of nuclear receptors.

Active Transport, Cell Nucleus↗